System, method and apparatus for the detection of patient-borne fluorescing nanocrystals

ABSTRACT

System, method and apparatus wherein a probe employing non-imagining optics is utilized in conjunction with a fluorescing nanocrystal tracer at the body of a patient. Excitation components within the probe working end are utilized to excite the nanocrystals to fluoresce at wavelengths in the near infrared region, such fluorescent energy is homogenized by interacting with involved tissue to provide a uniform fluorescing intensity over the surface of a photo-detector. Initialization and background determination procedures are described along with a technique for determining statistically significant levels of fluorescing activity.

CROSS-REFERENCE TO RELATED APPLICATIONS STATEMENT REGARDING FEDERALLYSPONSORED RESEARCH

Not applicable.

BACKGROUND OF THE INVENTION

In about 1983, a surgical oncologist, E. W. Martin, Jr., M.D. and aphysicist-electrical engineer, M. O. Thurston, Ph.D. combined theirtalents to explore then perceived deficiencies in the treatment ofcolorectal cancer. Why did it recur? Investigators then opined that sometumor was “missed” in surgery in that it was not located bypre-operational imaging, intra-operative visualization or palpation.Such hidden neoplastic tissue was referred to as “occult” tumor and wasconsidered to be an important aspect of the recurrence of cancer andlessoning of patient survivability. The Martin-Thurston approach at thattime was to radio-label an antibody specific to the tumor, inject itprior to surgery, then carry out surgery using current surgicalprotocols, whereupon a hand-held probe radiation detector was usedintra-operatively to scan the surgical site for occult tumors. Earlystudies showed the necessity of delaying this surgery following theinjection of locator or radiolabeled antibodies. That delay permittedthe labeled antibody to preferentially concentrate at neoplastic tissueas well as to permit normal body function based reduction of backgroundradiation. This procedure is described in U.S. Pat. No. 4,782,840 byMartin, Jr. and Thurston entitled “Method for Locating, Differentiating,and Removing Neoplasms”, issued Nov. 8, 1988. The procedure was referredto as “radio immunoguided surgery” or “RIGS”.

The evolution of the hand-held probe and its associated control systemwas not a trivial endeavor, requiring extensive research. In thisregard, the system was required to distinguish the totally random andspontaneous isotopic emissions of occult tumor from the same form oftotally random isotopic background emissions. For a variety of importantreasons, ¹²⁵I was elected as the radioisotope label of choice, having ahalf-life of about sixty days. Concerning the randomness of an isotope,from a given point in time, an atom of ¹²⁵I might emit a photon afterfive minutes from a starting point and another omission might occur ayear later. The sixty day half-life is an average (the time required forthe rate of such emissions to decrease to one half). A successfulcontrol approach for differentiating neoplastic tissue from backgroundwas derived which was statistically based upon count rate. The basis ofthat statistical control is described by Ramsey and Thurston in U.S.Pat. No. 4,889,991 entitled “Gamma Radiation Detector with EnhancedSignal Treatment”, issued Dec. 26, 1989.

Returning to the ¹²⁵I radiolabel, studies carried out by Dr. Thurstonshowed that to accommodate the RIGS procedure with the necessarygenerally multi-week waiting period permitting body clearance ofbackground radiation, a relatively longer half-life isotope having nohigh energy component and a dominant low energy was called for. ¹²⁵I wasessentially the only isotope with characteristics suitable for the RIGSprocedure. However, both patients and health care personnel were notentirely receptive to working with or being injected with thisradioactivity. While the sixty-day half-life of ¹²⁵I fulfilled thewaiting interval needs, it posed problems among others, with respect toregulatory agency requirements. Radioactive material, including anythingcontaminated with it must be protectively stored for a period amountingto ten times its half-life. Thus, for the case of ¹²⁵I, the storageinterval became six hundred days, a time element considered quiteburdensome. Notwithstanding its lower energy characteristic (27 Kev) theemissions from this labeling isotope could inflict damage uponassociated antibodies during shelf life. Thus about one out of each sixantibodies were labeled.

During the formative years of the RIGS system, the locators orantibodies, which were radiolabeled, were specific to the neoplasm ortumor itself. As these materials were improved, the locators developedwere specific to tumor-associated cell surface antigens. The term “cellsurface antigen” refers to an antigen of the plasma membrane proper andto any part of the tumor cell periphery, including the extracellularmatrix. Most of the antigens demonstrated on the surface of cells havebeen chemically defined as polysaccharides, glycoproteins, glycolipidsor proteins. A high molecular weight (200,000-400,000) tumor associatedglycoprotein, called TAG-72, is present in 85% of colorectal cancersalthough there is considerable heterogeneity in its expression in theprimary tumor, lymph nodes, and distant metastasizes. TAG-72 occurswidely on human carcinoma cells, including certain human breastcarcinoma cell lines, but is absent in normal healthy adult tissues,except secretory-phase endometrium. One of the first antibodies of thistype used with the RIGS system was called B72.3.

Colorectal adenocarcinomas have their genesis in mucin-secreting cells.Colon cancer occurs in the lining of the colon, which has to be coatedat a rather high rate with mucin both for lubrication and digestivejuice protection purposes. The epithelial cells involved reproduce at avery high rate. When such cells transform to cancer cells, they continueto secrete mucin but such mucin is distinctly different from mucinproduced from non-malignant cells. It was found that TAG antibodies werecapable of binding to these abnormal mucins called sialomucins, tolocate or indicate the presence of cancer cells.

A variety of monoclonal antibodies reactive with human gastrointestinalcarcinoma evolved. Of particular note, were the monoclonal antibodies(MAb) CC49(ATCC CRL9459) and CC83 (ATCC CRL9453) developed by Schlom andcoworkers at the National Cancer Institute. These antibodies exhibitincreased reactivity to antigen-positive tissue, reflecting a higheraffinity. See U.S. Pat. No. 5,512,443.

Essentially hundreds of surgical procedures were carried out by surgeonsemploying the RIGS system in conjunction with sialomucin bindinglocators, such as CC49 and CC83. During this period E. W. Martin, Jr.and associating surgeons detected “probe positive” (locator boundsialomucin) lymph nodes. It may be recalled that the locators arespecific to a by-product of tumor as opposed to tumor itself. Generally,these lymph nodes were readily accessible with the RIGS probe, beingpresent along, for example, the aorta, vena cava or near the liver.Notwithstanding the controversy necessarily involved in removing lymphstructure, these probe-positive lymph nodes were directed to pathologyalong with the resected tumor burden. The generally received responsewas that the dissected nodes were hyperactive but no presence of cancercells was detected. The issue as to the appropriateness of removingthese probe-positive lymph nodes remained essentially until patientsurvival data was evolved. Data collected with respect to patientshaving had probe-positive lymph nodes removed indicated what has beencalled “remarkable” survival improvement. An analysis was subsequentlycarried out with respect to the question as to whether a small number ofmalignant cells in a lymph node would be readily detected by frozensection techniques. Assuming a spherical node of 0.5 cm diameter, anexamination of the entire node in six-micron sections would require over800 sections with a total area of 1600 cm². The likelihood of observingmalignant cells would have been, at best, remote. See the followingpublication:

-   -   1. Barbera-Guillen, et al., “First Results for Resetting the        Anti-Tumor Immune Response by Immune Corrective Surgery in Colon        Cancer” Am. J. Surg, 1998, 176:339-343.

In the early 1990s investigators utilized the RIGS system to locate,differentiate and stage other types of cancer, for instance, endocrinetumors involved, inter alia, with breast, children, gastrinomas, lungand nervous system. Generally, the approach was to administer aradiolabeled somatostatin congener to assess the patient with the RIGSprobe. However, before subjecting the patient to such administration, aninitial determination preferably was made as to whether the radiolabeledsomatostatin congener would bind to the tumor site, i.e., whethersomotostatin receptors are associated with the neoplastic tissue. Thiswas conveniently done with a wide variety of endocrine tumors, whichrelease peptides or hormones, referred to as “biochemical markers.” Inorder to make this determination, initially a biochemicalmarker-inhibiting dose of unlabeled somotastatin congener wasadministered to the patient. The biochemical marker associated with theneoplastic tissue then was monitored to determine whether theadministered samotostatin congener reduces the presence of the marker inthe patient. If the monitored presence of the marker was reduced, thenthe surgeon could be confident that the neoplastic tissue or tumorcontains receptors to which the somatostatin would bind. Thus, theadministration of radiolabeled somatostatin congener was appropriate forsuch patient. If the biochemical marker associated with the neoplastictissue was not appropriately reduced following the administration of theunlabeled somatostatin congener, then the neoplastic tissue may not bedeterminable by the use of radiolabeled somatostatin congener andalternative modalities of treatment would be considered, such as the useof radiolabeled antibodies.

See: O'Dorisio, et al., U.S. Pat. No. 5,590,656; entitled “Applicationof Peptide/Cell Receptor Kinetics Utilizing Radiolabeled SomatostatinCongeners in the In Situ, In Vivo Detection and Differentiation ofNeoplastic Tissue”; issued Jan. 7, 1997 and incorporated herein byreference.

For a variety of reasons, the use of the RIGS system was suspended andoncologists have no technique available for detecting cancer inducinglymph nodes. In consequence, the survival rates for patients havingundergone tumor-burden removal associated with colonic cancers havegenerally descended to pre-RIGS levels.

BRIEF SUMMARY OF THE INVENTION

The present disclosure is addressed to system, method and apparatuswherein an intra-operative probe structured with non-imaging optics isemployed in concert with a locator incorporating a fluorescingnanocrystal and which specifically binds a marker produced by orassociated with neoplastic tumor. With the method, lymph tissueassociated with neoplastic tissue becomes detectible and thus removeableopening an opportunity to return to the patient survival rates achievedwith the earlier RIGS system.

The probe apparatus, structured with non-imaging objects is capable ofmedical uses beyond cancer therapy. A working end of the probe isconfigured having a photo-detector such as a photodiode with anassociated pre-amplification stage, a longpass filter functioning toblock excitation and ambient wavelengths, and an encapsulating assemblywith a forwardly disposed transparent window having a forwardtransmission surface contactable with tissue. Because near infraredreturning fluorescing energy is scattered by tissue, its intensitybecomes uniform or is homogenized to achieve reliable intensity-baseddetector output.

The system at hand is one wherein the operator is prompted from acontrol console display to carry out necessary initialization proceduresinvolving a small dark chamber created by a cap extendable over theprobe working end. This cap is configured with an internal forwardsurface formed of a tissue emulating polymeric material. As a firstinitialization procedure the cap is positioned over the probe workingend, the photo-detector is enabled and dark current/electronic noise isdetected and its value is stored. A second initialization then ensueswith the energization of the excitation (LED) components and enablementof the photo-detector to measure the intensity of back scatteredexcitation illumination which will be in the red region of the spectrum.The sum of these photo-detector values both during initialization andsubsequent scanning becomes a stored reference value which is subtractedfrom scan values. Such control is dependent upon the type of fluorescingnanocrystal being used, the photo-detector being enabled insynchronization with the energization of the excitation components for aType I nanocrystal. Where a Type II nanocrystal is used there is arandom delay between the time of excitation of a crystal and thesubsequent emission of a fluorescence photon. For a large number ofnanocrystals the rate of emission is a decreasing exponential functionof the time after excitation with a time constant of the order of amicrosecond. For this type fluorescing nanocrystal the photo-detector isenabled for a sampling interval subsequent to the interval of excitationand total fluorescing intensity is computed, for example, by aconvolution procedure.

Another initialization procedure which, in effect, is a calibrationapproach also is carried out with a light-type cap carrying a Type I orType II fluorescing nanocrystal material. With the cap in place, theexcitation components are energized and the photo-detector is enabled inappropriate sequence to obtain an intensity readout. That readout thenis compared by the operator with a readout value located on the outsideof the cap.

When the system is employed in conjunction with a locator whichincorporates a fluorescing nanocrystal and specifically binds a markerproduced or associated with neoplastic tissue, the patient is injectedwith such locator and a clearance interval ensues. Typically thatclearance interval will be about two to three weeks to permit asubstantial diminution of that locator which does not bind a marker. Atthe time of surgery, the probe is moved across normal tissue to obtain abackground level of fluorescence. The system then computes astatistically significant threshold above mean background to determinewhen the transmission surface of the probe is over cancer involvedtissue. In general, the threshold will be three standard deviations overmean background, such a three sigma value being selected because theexpected probability of a false positive reading would be less than 1%.

The probe-based system may be employed with a generally cylindrical handgraspable support extending to an angularly disposed working end.Additionally, the system may be employed with a finger mounted supportwhich slides over a finger of a surgeon. A third support is intended forlaparoscopic surgery wherein the working end components are mounted soas to be “side looking” and supported from an elongate accessing tube.

Another object of the disclosure is to provide a method for the surgicaltreatment of patients afflicted with neoplastic tissue which comprisesthe steps:

-   -   (a) administering to a patient an effective amount of a locator        incorporating a fluorescing nanocrystal and which specifically        binds a marker produced by or associated with neoplastic tissue;    -   (b) permitting time to elapse following step (a) for the locator        to preferentially concentrate at any marker and for unbound        locator to be cleared so as to increase the ratio of        fluorescing-based radiation from specifically bound locator to        fluorescing-based radiation representing background in the        patient;    -   (c) after the clearing time has elapsed in step (b) surgically        accessing an operative field of the patient;    -   (d) providing a hand manipulative probe having forwardly        disposed excitation components energizable to cause any        nanocrystal to fluoresce at a detection wavelength or        wavelengths and a forwardly disposed photo-detector configured        for response to non-imaged detection wavelengths so as to        develop detection outputs corresponding with fluorescing        radiation intensity;    -   (e) using the probe, determining and storing the fluorescing        radiation intensity at the background;    -   (f) based upon the fluorescing radiation intensity at the        background, determining a statistically significant fluorescent        radiation intensity value whereat a perceptible cue is        generated;    -   (g) removing tumor burden from the patient using the probe where        necessary to locate and differentiate neoplastic tissue; and    -   (h) using the probe, determine and remove lymph tissue sites        exhibiting detection outputs.

Other objects of the disclosure will, in part, be obvious and will, inpart, appear hereinafter. The invention accordingly, comprises thesystem, apparatus and method possessing the construction, combination ofelements, arrangement of parts and steps which are exemplified in thefollowing detail description.

For a fuller understanding of the nature and objects hereof, referenceshould be made to the following detailed description taken in connectionwith the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a colon cancer patient survival probability chart resultingfrom the utilization of prior radio immuno-guided surgery with respectto stages I through IV;

FIG. 2 is a chart similar to FIG. 1 but showing colon cancer patientsurvival probability for stages I and II only;

FIG. 3 is a colon cancer patient survival probability chart similar toFIG. 1 but including only patients at stages III and IV;

FIG. 4A is a block diagram showing the functional components of a probesuited for intra-operative use;

FIG. 4B is an extension of FIG. 4A and illustrates in block diagrammaticfashion the functional components of a control assembly;

FIG. 5 is a perspective view of the present system showing a controlassembly console, probe and associated cable;

FIG. 6 is a sectional view of an probe taken through the plane 6-6 shownin FIG. 6;

FIG. 6A is an enlarged partial view of the working end of the probe ofFIG. 6;

FIG. 7 is a front view of the working end of the probe of FIG. 6;

FIG. 8 is a perspective view of an initialization cap employed with thesystem of the invention;

FIG. 9 is a sectional view taken through the plane 9-9 shown in FIG. 8;

FIG. 10 is flow chart illustrating an initialization procedure;

FIG. 11 is a flow chart illustrating an initialization procedureproviding a calibration check of the system;

FIG. 12 is a flow chart illustrating a scan routine;

FIG. 13 is a timing diagram illustrating a pulse-based procedure forutilization with Type I nanocrystal;

FIG. 14 is a timing diagram showing a pulse-based method utilized inconjunction with Type II nanocrystal;

FIG. 15 is a perspective view of a surgeon's hand, arm and shouldershowing the mounting of a finger supported intra-operative probe;

FIG. 16 is a front view of a surgeon's hand showing an intra-operativeprobe which is finger mounted;

FIG. 17 is a perspective view of a finger-supported intra-operativeprobe;

FIG. 18 is a sectional view taken through the plane 18-18 shown in FIG.17;

FIG. 19 is a perspective view of a probe configured for laparoscopicutilization;

FIG. 20 is a sectional view taken through the plane 20-20 in FIG. 19;and

FIG. 21 is a top view of the working end of the probe of FIG. 19.

DETAILED DESCRIPTION OF THE INVENTION

In the discourse to follow, three charts initially are presentedillustrating about ten year survival probability data with respect tocolorectal cancer patients having undergone surgery which utilized theRIGS procedure. The discussion then turns to nanocrystals which may beexcited to fluoresce and function to supplant the radiolabel employedwith the RIGS procedure. In this regard, the instant system utilizingsuch nanocrystal technology then is described in conjunction with blockdiagrams, an illustration of the working end of a probe and a systeminvolving a probe, control assembly console and initialization chambersor caps. Flow charts concerning two initialization routines and scanroutines are then described in conjunction with two modulation timingdiagrams concerning two nanocrystal implementations. Lastly,finger-mounted and laparoscopic probe structures are illustrated anddescribed.

To gain an insight into the utilization of cancer detection systems suchas the earlier-described RIGS approach, reference is made to FIG. 1which plots the survival probability against time in months for apopulation of 83 patients who had undergone surgery using the RIGSapproach. The plots, in effect, represent life expectancy, drops in thecurves being the result of the death of patients from any cause. Forexample, in addition to cancer occasioned deaths, deaths could be fromheart attacks, strokes, auto accidents and the like. The data includesall Stages I through IV of the cancer involvement. In this regard, StageI involves an early and preliminary detection of cancer and as thestages increase in number, cancer involvement becomes more extendeduntil Stage IV which fundamentally is considered a terminal condition.Where the RIGS system indicated that all cancer involved tissueincluding that in the lymph system was removed, then the cases areconsidered to be “RIGS negative” and their survival probability isrepresented at curve 10. On the other hand, there were conditionswherein the RIGS probe would show a continued presence of cancer butunder conditions, for example, involving vital tissue where allindications of cancer could not be removed and the condition wasconsidered to be “RIGS positive”. This RIGS positive condition isplotted at curve 12. The median survival in months with respect to curve12 for RIGS positive patients was 26.0. Correspondently, the mediansurvival in months for RIGS negative patients as represented at curve 10was 106.5 months or about 8.9 years. The probability factor, p, forcurves 10 and 12 was 0.001 representing a statistical reliability ofabout 1 in 1000 which is an excellent value. The curves further showthat five year survival was 29% with respect to RIGS positive patientsand 70% with respect to RIGS negative patients. At 10 years, curve 12reveals the probability of 21% survival with respect to RIGS positivepatients and curve 10 represents a 10year probability of survival of 49%with respect to RIGS negative patients.

Turning to FIG. 2, similar survival probability data is charted but withrespect to Stages I-II only. Curve 14 charts survival probability forpatients deemed RIGS negative with respect to months, while curve 16plots survival probability for patients having been deemed RIGS positivewhere RIGS identified cancer involvement remained after surgery. Themedian probability of survival with respect to curve 16 and RIGSpositive patients was 43.1 months, while the median probability inmonths for the survival of RIGS negative patients was 151.1 months orabout 12½ years. As before, the deaths could be for any cause but cancerdeaths are included. The five year probability of survival with respectto curve 16 was 41% and the corresponding five year survival withrespect to curve 14 was 79% At ten years the probability of survivalwith respect to curve 16 was 30% and by contrast, the probability ofsurvival with respect to curve 14 was 55%. The probability factor, p,for curves 14 and 16 was 0.023 which remains a good value.

Lastly, looking to FIG. 3, a similar chart is provided but in this case,with respect to patients having been deemed at Stages III or IV. Curve20 represents that the median number of months probability of survivalwas 20.0 while, correspondently, the mean probability for survival ofpatients represented at curve 18 was 75.9 months or about 6.3 years. Thefive year probability of survival with respect to curve 20 was 16% andthe corresponding five year survival probability for curve 18 was 58%.At ten years, the probability of survival from deaths of any cause asrepresented at curve 20 was 5% and the corresponding ten yearprobability for survival with respect to curve 18 was 42%. Theprobability factor, p, for curves 18 and 20 was 0.001. As is apparent,providing a viable intra-operative cancer detection technique will bemost beneficial.

Semiconductor nanocrystals are gaining popularity due to the ability ofindustry to tailor their optical and energetic properties by varying thesize, shape and material parameters. Quantum confinement of chargedcarriers in nanocrystals to sizes comparable to their excitonic Bohrradius results in discrete energy levels and narrow optical transitions.Nanocrystals enjoy a broad range of applications ranging from biologicalimaging, quantum computing and solar cells to electroluminescentdevices.

Two types of nanocrystals are incorporated with the instant system. Afirst type employs fluorescent nanocrystals such as CdSe, CdTe (CdX).With these fluorescent nanocrystals, the emission of a photon is delayedfrom the time of absorption of an excitation photon by a random timeinterval of about 10nanoseconds. The crystals are designed to fluorescein the near infrared region of the spectrum to enhance transmissionthrough tissue. In this same regard, the nanocrystals are excited byphotons at a higher energy level, i.e., photons with a shorterwavelength. In general, again looking to transmission through tissue,excitation energy preferably will be in the red region of the spectrum.The nanocrystals are arbitrarily designated herein as a Type I. Adiscussion of water-soluble functionalized nanocrystals of the CdXvariety is set forth in U. S. Pat. No. 6,114,038 by Castro, et al.,entitled “Functionalized Nanocrystals and Their Use in DetectionSystems”, issued Sep. 5, 2000, and incorporated herein by reference.Reference also is made to the following publication:

-   -   2. Castro, S., “Biopixtal Nanocrystalline Fluorescent Markers”,        Genetic Engineering News, Vol 19, No. 17, Oct. 1, 1999.

A second approach for developing a fluorescing locator is through theutilization of a family of PbS nanocrystals that are coated with a thinlayer of a dielectric compound. When a photon of excitation is absorbedby a this type fluorescent nanocrystal the emitted photon has a randomdelay constraint or time that is of the order of a microsecond.Accordingly, where there is a modulation of the excitation energy, forexample, in the form of short pulses, the fluorescence will continue inexponential decaying fashion after the excitation has stopped. To usethis effect, the reading or sampling pulse is started a short time afterthe excitation pulse ends and a total fluorescing intensity is computedwith a convolution approach. These nanocrystals are arbitrarilydesignated herein as a Type II. See the following publication:

-   -   3. Warner, et al., “Time-Resolved Photoluminescence Spectroscopy        of Ligand-Capped PbS Nanocrystals”, NanoTechnology 16 (2005)        175-179.

The use of excitation energy in the red region of the spectrum incombination with fluorescence in the near infrared region is attractiveparticularly with respect to identifying lymph nodes retaining locatorsin the course of colorectal surgery. These nodes are relatively small,generally only about 3 mm in diameter and they are close to the surface.Accordingly, the spectrum at hand is one capable of being useable atdepths of 2 cm and 3 cm which is entirely adequate. Studies undertakenduring the years of utilization of the RIGS system developed criteria ofsensitivity for detecting such lymph nodes. Investigators were able tocalculate the number of atoms of radioactive iodine which were in thelymph nodes. It turned out that the nodes contained about 300 millionatoms of iodine and associated antibodies. For practical reasons, thenumber of antibodies was greater than the number of iodine atoms andaffected lymph nodes showed a RIGS or isotopic radiation count of about10 counts per second over background, a condition requiring rathersignificant statistical analysis. However, this situation alters withthe use of nanocrystals fluorescing in the near infrared region. In thisregard, about 300 million or so small fluorescent nanocrystals will belocated in a node, all of which may be excited repeatedly. By contrast,once an iodine atom has radiated, it becomes tellurium and no longerusable in the detection process. With the nanocrystal approach,theoretically, with respect to a given affected lymph node, a billionphotons per second may be generated. The instant system, may, forexample, excite at a shorter wavelength of about 650 nanometers toachieve a nanocrystal fluorescence in the order of 700 nanometerwavelengths. For both excitation and fluorescing response there will besome degree of reduction of signal strength. This reduction comes aboutfrom the absorption of radiation by tissue. In this regard, suchabsorption primarily will be a result of the water content of the tissueand is not as significant as the scattering of photons as they passthrough or impinge upon tissue. Such scattering results in a reductionof signal strength and of blurring, the latter result being advantageousinasmuch as the receiving optics are non-imaging, in effect, aLambertion radiation intensity distribution being desirable over theface of a photo detector.

See the following publication with respect to non-imaging optics:

-   -   4. Winsron, et al., “Non-Imaging Optics”, Elsevier Academic        Press, Boston Mass., 2005.

By employing a non-imaging optical system as opposed to an imaging one,the surface of a photo-detector will receive fluorescing photonradiation which is both directly confrontational but also evolves fromvery shallow angles occasioned by significant scattering in tissue.Sensitivity is substantially improved over an imaging optical system.

FIGS. 4A and 4B combine to respectively represent the probe component ofthe system and a control console component of the system at hand.Looking to FIG. 4A, the functional components of the probe arerepresented in general at 30, the probe incorporating a working end, thecomponents of which are represented in general at 32 and a support, thecomponents of which are represented in general at 34. Looking to workingend 32, a photo-detector, for example, a photodiode is represented atblock 36 which performs in conjunction with an integrating preamplifieras represented at arrow 38 and block 40. Photodiode 36 may be provided,for example, as a type SD200-11-31-241, marketed by Advanced Photonix.Such devices may be obtained with an integrally retained preamplifier.Photodiode 36 is isolated from most of the ambient illumination whichmay be encountered in an operating room theater as well as excitationwavelengths by a longpass filter represented at 42. Operating rooms aretypically structured with very intense incandescent lighting which willexhibit an infrared component. Filter 42 is intended to blocksubstantially all (excitation and ambient) but those components whichmay be accommodated for by initialization and modulation approaches togenerating excitation illumination and enabling photodiode 36. Extendingaround the longpass filter 42 is an excitation assembly 44 including amount and, for instance, an array of eight light emitting diodes whichmay be provided, for instance, as light emitting diodes emitting in thered region of the spectrum (Lumex Surface Mount LED Digkey Part#67-1727-1-ND). These excitation components are illuminated from anexcitation driver network represented at block 46 as indicated by arrow48. Securing the working end from body fluid contamination is a verythin transparent window represented at 50. The random, essentiallyhomogenized fluorescing illumination is represented by the randomlyoriented arrow array identified generally at 52. Preferably theexcitation diodes are canted slightly inwardly to effect a red regionillumination convergence. Such directionality is represented by arrows54 and 56. Excitation driver 46 may be located within support portion 34of probe 30 along with such components as an optional local or probecontrol processor represented at block 58. Processor 58 is under thecontrol of a main processor which is console mounted and such control isrepresented at arrow 60. Local controller 58, as represented at arrow 62asserts control over a switching controller represented at block 64 asrepresented at arrows 66 and 68. Controller 64 functions to enablephotodiode 36 as well as integrating preamplifier 40. The output of theintegrating preamplifier is directed to the console mounted mainprocessor as represented at arrow 70. Controller 64 additionally assertsmodulating control over the excitation driver 46 as represented at arrow72.

Where the probe structure permits, the support 34 may incorporate twoswitches which are finger actuatable by the surgeon. In this regard, thesupport portion 34 may incorporate a background switch represented atblock 74 which provides an input to the processor 58 as represented atarrow 76 as well as to the main processor as represented at arrow 78. Byactuating switching 74, the amount of background fluorescing radiationwhich is not cancer involved may be assessed and from that information astatistically significant threshold fluorescing intensity level may becomputed. The second switch which may be mounted at the support 34 is arecord switch as represented at block 80. Switch function 80 provides aswitching signal directly to the main processor as represented at arrow82. If actuated or held down for a short interval, the switch will causea voice audio annunciation of an intensity level. Should switch 80 beheld down for a longer interval, then a microphone is activated topermit the surgeon to comment as to the location, for example, offluorescing nanocrystal material which has specifically bound a markerproduced by or associated with neoplastic tissue. When such tissue isidentified, the main processor will provide a cue which preferably isaudible. Such a cue may also be generated at the probe support 34itself. For example, a visual cue may be provided by energizing a lightemitting diode as represented at arrow 84 and block 86.

Referring to FIG. 4B, the functional components of a control assemblyassociated with probe 30 are represented in block form. In the figure,arrows 70, 82, 78 and 60 reappear from FIG. 4A and are represented asbeing a component of an elongate flexible cable identified generally asat 92. These components of cable 92 extend to a main processorrepresented at block 94. Processor 94 performs interactively with flashmemory represented at block 96, the interactive function beingrepresented at arrows 98 and 100. Device 94 may be provided as a typeSTR 755FVO marketed by S T Microsystems, Inc., of Geneva, Switzerland.As noted above, it may also perform the function of probe processor 58.In general, processor 94 will respond to an analog input as indicated atarrow 70 representing data derived from the integrating preamplifier 40(FIG. 4A). That data will be digitized and evaluated with respect to theprocedure being carried out. In this regard, during a scan mode ofoperation the incoming data will represent the intensity offluorescence. That intensity will be statistically evaluated againstmeasured background fluorescence developed from a scan of tissue whereinlocator is not preferentially present. Where a statistically significantamount of evaluating fluorescent intensity is present, then asrepresented at arrow 102 and block 104, a numerical data signal isderived. As represented at arrow 106 and block 108, the signal may bedirected to an audio switch under the control of processor 94 asrepresented at arrow 110. The signal at arrow 106 will then betransmitted as represented at arrow 112 to an amplifier represented atblock 114. Amplifier 114 provides an audio signal to a loudspeaker 116as represented at arrow 118. Such a signal audibly alerts the surgeonthat the forward surface of the probe is over sufficient fluorescinglocator which will have specifically bound a marker produced by orassociated with neoplastic tissue. During the era of the use of the RIGSsystem, the sound was referrer to as a “siren”. The surgeon also maywish to hear a voice annunciation of the numeric level of intensity offluorescence which the probe is encountering. This can be carried out,for example, by actuating the record switch described at block 80 inconnection with FIG. 4A. Note in this regard, the main processor is seento be operationally associated with a speech generator as at block 120as represented at arrow 122. As represented at arrow 124, the voiceoutput of generator 120 is directed to audio switch 108, again undercontrol of the main processor 94 as represented at arrow 110. The voiceinformation then is directed to amplifier 114 as represented at arrow112 and is outputted to loud-speaker 116 as represented at arrow 118.The fluorescence intensity related numerical data also may be displayedat a display panel represented at block 130. In this regard, mainprocessor 94 is associated with an input/output (I/O) functionrepresented at block 132 as indicated by arrows 134 and 136. Port 132 isshown operationally associated with display panel 130 by arrow 138. I/Oport 132 also may be operationally associated with a keyboard input asrepresented at block 140 and arrow 142. It is also advantageous that themain processor 94 be capable of communication with a remote computer.Such an optional remote computer is represented at block 144 and theinteraction with that computer via port 132 is represented at arrows 146and 148.

Finally, power is supplied to the control assembly 90 as represented byan a.c. source 156. As represented at arrow 158 and block 160, the a.c.source is directed to an isolation transformer and, in turn, asrepresented at arrow 162 and block 164 to control assembly power supply.

Referring to FIG. 5, a perspective view of the instant system ispresented and identified in general at 170. Functions described in FIGS.4A and 4B are identified in the instant figure with the same numeration.In this regard, control assembly 90 is identified in conjunction with aconsole 172 having a relatively large liquid crystal display againidentified at 130. Display 130 provides numerical read-out offluorescing intensities as well as a cueing to the operator particularlywith respect to initialization procedures. Next to the display 130 is anaudio grill 172 behind which a loudspeaker as earlier-described at 116may be located. Cable 92 extends from a cable connector 174 to a probeagain identified in general at 30 having a working end 32 and a support34. Console 172 also is configured with a number of switches includingan on/off toggle switch 176; record switch 178; a “squelch” switch 180;a calibration switch 182; an initialization 1 switch 184; aninitialization 2 switch 186 and a two-position toggle switch 188 whichfunctions to introduce the type of fluorescing nanocrystal employed,either a Type I or Type II. The term “squelch” as used herein becamepopular with practitioners of the earlier RIGS system and has a meaningsomewhat different than given, for instance, in the authoritativedictionary of IEEE Standards Terms, 7^(th) Addition which gives the termtwo meanings (1) a circuit function that acts to suppress the audiooutput of a receiver when noise power that exceeds a predetermine levelis present; or (2) facility incorporated in radio receivers to disabletheir signal output while the received carrier level is less than apreset value. In contrast, the squelch switch 180 is employed to developa statistical fluorescing intensity value based upon a measuredbackground fluorescing value. For example, with the instant procedure,the probe instrument 30 initially is positioned and scanned in thevicinity of a region not involved with cancer, for example, in thevicinity of the heart or aorta in order to obtain a blood poolbackground fluorescing intensity. The microprocessor 94 then calculatesa statistically significant value, for example, a predetermined numberof standard deviations of the mean background fluorescing intensity toderive a statistically significant threshold radiation count rate level.

In general, the console 172 will be located outside of the sterile fieldwithin a surgical theatre, while the probe instrument 30 will be withinthat sterile field and in the hand of a surgeon. As discussed inconnection with FIG. 4A, it will be beneficial to afford the surgeon theopportunity to hand actuate the functions of switch 180 as described at74 and switch 178 as described at 80 in FIG. 4A from the probe itself.The figure shows that the probe instrument 30 is configured having agenerally cylindrical unitary housing 190 (support 34) which is handgraspable by the surgeon and is interactively associated with theconsole 172 via cable 92. Other forms of interactive transmission whichare wireless may be employed in place of a cable as shown. The handgraspable support or unitary housing 190 extends from a forward end 192and a rearward end 194. The term “unitary” is used herein to indicatethat no joints or unions are present in the housing to establish aswitching function. A working end 32 is coupled to the forward end 192of housing 190 while the cable 92 is coupled at the rearward end 194.Two planar switch actuating surfaces are formed integrally into thehousing 190 as shown at 196 and 198. Switches 196 and 198 carry out thefunctions described earlier respectively at 74 and 80. In this regard,surface 196 carries out a background or squelching function and thesurface 198 carries out the above-discussed recording functions. Lookingadditionally to FIG. 6, it may be observed that surfaces 196 and 198 aremachined into the housing 190 in a manner providing forwardly andrearwardly disposed bevels shown respectively at 200 and 202 whichfunction, with the surfaces 196 and 198 to define a switching region204. Intermediate the surfaces 196 and 198, a beveled rib 206 is definedhaving a flat, upwardly disposed surface 208 establishing a rib heightwhich falls below the external periphery of housing 190. The thusdefined switch region 204 is readily tactilely identifiable to thesurgeon such that surface 196, carrying out the background function orsquelch function of switch 180 is easily determined by the surgeon withrespect to rib 206 and bevel 200. Similarly, the surgeon readilytactilely identifies the switch actuating surface 198 such that thefunctions of record switch 184 are easily determined by the surgeon withrespect to rib 206 and bevel 202.

FIG. 6 reveals that the unitary housing 190 is configured having aninternally disposed switch-receiving channel 210 which is open andaccessible at the rearward portion of housing 190 through a cylindricalbore-formed cavity 212 as well as from a cylindrical bore-formed cavityof shorter length at the forward end as seen at 214. Channel 210 isaccurately formed utilizing, for example, a wire electrical dischargemachine (EDM). This permits a very accurate formation of an upwardlydisposed switch contact surface 216 and a parallel planar oppositelydisposed load surface 218. Each of the surfaces 216 and 218 and switchcontact surface 216 is parallel with and spaced from switch actuatingsurfaces 196 and 198. A predetermined distance defining a switch wall220 of thickness selected such that flexure at surfaces 196 and 198under finger pressure is so minor as to be tactilely undetectable. For apreferred aluminum housing 190, that thickness will range from about 15mils to 20 mils and resultant flexure upon switch actuation fromsurfaces 196 and 198 will be in a micro-inch range such that, in effect,the operator is transmitting hand generated stress with almost noaccompanying material strain. Accordingly, sealing is achieved due tothe unitary structure of the construction of housing 190 without theimposition of fatigue which otherwise might evoke the presence of cracksat switching region 204 to thus permit the ingress of body fluids intothe internal regions of the probe instrument.

Positioned in abutting adjacency with switch contact surface 216 is thepressure responsive surface of a two-component thin piezoelectric switch222. Such pressure responsive surface of the two-component switch 222 issupported from a stiff substrate, for example, formed of FR4 material.The bottom of this material is a flat oppositely disposed supportsurface which incorporates three terminals (not shown). With thearrangement, one switching component is located directly beneath switchactuating surface 196 and the other directly beneath switch actuatingsurface 198. Switches as at 222 are marketed by Wilson-Hurd, Inc., ofWasusau, Wis. and have been described, for example, in U.S. Pat. No.4,857,887, issued Aug. 15, 1989. Preferably, such switches are preloadedin compression to enhance their performance.

To retain switch 222 compressively against the switch contact surface216, a switch support assembly shown generally at 224 is provided.Assembly 224 is formed of two complimentary wedges 226 and 228 formed ofaluminum with matching sloping surfaces which serve to provideoppositely disposed parallel outer surfaces between the load surface 218and the bottom of switch 222. Transmission leads extend from the switch222 to a printed circuit board 230 structurally and operationallysupporting the components described in FIG. 4A. Circuit board 230 isalso operationally coupled with cable 92 as represented at lead array232. The switching structure shown was developed for utilization withthe earlier-described RIGS system and is described at a higher level ofdetail in U.S. Pat. No. 5,682,888 by Olson and Thurston, entitled“Apparatus and System for Detecting and Locating Photon Emissions WithRemote Switch Control”, issued Nov. 4, 1997and incorporated herein byreference.

Circuit board 230 also supports the earlier-described LED function 86.In this regard, an LED is represented at 234 positioned beneath atransparent polycarbonate plug 236 mounted within housing 190. Rearwardend 194 of the housing 190 is connected to the necked down cylindricalportion 238 of a cylindrical rear cap 240. Rear cap 240 is intimatelycoupled with an elongate conically-shaped relief component 242 formed ofa medical grade silicone which surmounts and seals against cable 92.

Forward end 192 of the probe support 34 is canted at an angle of 15°with respect to probe axis 250. Attached to this forward end 192 is ashort connector tube 252, the rearward end of which also is canted at a15° angle with respect to axis 250. Connector tube 252 is connected tothe housing 190 at end 192 to provide a desirable 30° cant for theworking end 32. Looking additionally to FIG. 6A, an aluminum cylindricalcap 254 is attached to and extends over the forward portion of connectortube 252 and is seen to retain a transparent thin window 256 formed, forexample, of polycarbonate with a thickness of 0.020 inch. FIG. 6Areveals that cap 254 is positioned to over and connected to acylindrical extension with forward portion 258 of connector tube 252.Supported immediately against the interior surface of portion 258 is agenerally cylindrical copper excitation mount which has a forward edge262 which is inwardly beveled and functions to support an array ofexcitation diodes represented generally at 264. Looking additionally toFIG. 7, the array 264 is seen to comprise light emitting diodes 266a-266 h. These diodes emit in the red region of the spectrum and each issurface mounted upon a printed circuit supported by a thin polyimidesubstrate or film sold under the trade designation “Kapton”, by E. I.duPont de Nemours and Company. The printed circuit is represented inFIG. 6A at 268. The Kapton is adhesively secured to the forward edge 262of the excitation mount 260. Next inwardly from excitation mount 260 isa polymeric detection assembly mount 270. The outer surface of mount 270is notched to direct dual leads from the LED associated printed circuitmounts to circuit board 230 (FIG. 6). Two such leads are seen in FIG. 6Aat 272 a and 272 e. Mount 270 additionally is centrally bored to supporta pre-amplification stage again represented at 40; a forward detectoragain identified at 36 and a longpass filter again identified at 42.Filter 42 functions to block the red region excitation illumination fromthe LED array 264 as well as essentially all ambient illumination. Apreamplifier output is represented at lead pair 274 which also extendsto printed circuit board 230 (FIG. 6). In general, LEDs as at 266 a-266h will exhibit about a 2volt drop, thus, with an adequate power supply,permitting them to be coupled in series circuit fashion.

In the course of carrying out initialization procedures it is necessarythat the working end 32 of the probe be inserted within a light-tightchamber, preferably provided as a cup-shaped cap. Looking to FIG. 8,such a cylindrically shaped cap is identified in general at 280, theinternal cavity defined thereby being seen at 282. Turning to FIG. 9,cap 280 reappears in section and is seen to incorporate a polymericmaterial 284 which is structured to emulate the excitation lightscattering induced by human tissue. This particular cap is arbitrarilydesignated as cap A. An essentially identical cap, arbitrarilydesignated cap B will incorporate the fluorescing nanocrystal employedwith the system. One such nanocrystal will correspond to theabove-described Type I and a third cap, C, will incorporate nanocrystalsidentified above as Type II.

Referring to FIG. 10, a flow chart describing the initialization of thesystem in conjunction with cap A is set forth. Looking to that figure, asystem power-up is represented at symbol 290 and arrow 292. Thispower-up is carried out by actuating toggle switch 176 as described inconnection with FIG. 5. Under control of main processor 94 (FIG. 4B),the display 130 will prompt the operator to place cap A on the probe andpress “INIT1” (switch 184) when ready. Next, as represented at arrow 294and block 296, a query is made as to whether the INIT1 switch has beenpressed. In the event it has not, then the system dwells as representedat loop arrow 298. Where the INIT1 switch has been pressed, then asrepresented at arrow 300 and block 302, a display will provide a promptthat excitation pulses are off and the photodiode is on. Additionally itwill advise that the system will read an average, n, pulses and store aresultant value as DARK CURRENT. The resultant value representselectronic noise and dark current in the photodiode. With such storage,then as represented at arrow 304 and block 306, a prompt is published atdisplay 130 cueing the operator to press toggle switch 188 to elect aType I or Type II nanocrystal incorporating locator. Accordingly, asrepresented at arrow 308 and block 310, the operator enters nanocrystaldata, the election of Type I being represented at arrow 312 and thestorage of that information being represented at block 314.Correspondingly, as represented at arrow 316 and block 318 where a TypeII nanocrystal has been entered in the system via switch 188, then thesystem will store Type II.

Returning to block 314, where the nanocrystal at hand is Type I, then asrepresented at arrow 320 and block 322, the user is prompted to pressINIT2 button 186 while cap A remains in place. This will cause thesystem to turn-on excitation pulses and synchronizely enable or drivethe photo-detector 36. The result is a test of the longpass filter 42with respect to scattered excitation light. Accordingly, as representedat arrow 324, arrow 326 and block 328, the system reads and averages, n,pulses from the photodiode 36. That value then is added to the DARKCURRENT value described in connection with block 302 and the result isstored as REFERENCE.

Where a Type II nanocrystal has been stored, then as represented atarrow 330 and block 332, the excitation pulses are turned on. However,the system provides a delay in the photodiode read pulses until afterthe end of the excitation pulses. It should be kept in mind that this isa test of the accuracy of longpass filter 42 with respect to excitationlight. As represented at arrow 334, the system then carries out theprocedure represented at block 326. The initialization then continues asrepresented at arrow 336 and node A.

Referring to FIG. 11, node A reappears in conjunction with arrow 350extending to block 352. As represented at block 352, a prompt isprovided at display 130 calling for a calibration check to replace cap Awith either cap B or cap C. Additionally, the operator is instructed topress the calibration switch (switch 182) when ready. Next, asrepresented at arrow 354 and block 356, a determination is made as towhether console switch 182 has been pressed. In the event that it hasnot, then as represented at loop arrow 358, the system dwells. However,when the switch button has been pressed, as represented at arrow 360 andblock 362, the system integrates, n, photodiode pulses and subtractsREFERENCE. The result is displayed as a numerical fluorescence intensityvalue identified as “CALIBRATION VALUE”. That number is then compared bythe operator with a calibration number carried by cap B or cap C. Wherethose numbers are substantially the same, then the operator knows thatthe system is properly operating. As represented at arrow 364 and node366, the initialization procedure then is completed.

In general, with the procedure at hand, the patient is administered aneffective amount of a locator incorporating a fluorescing nanocrystaland which specifically binds a marker produced by or associated withneoplastic tissue. Administration is by injection. Following theinjection, a clearing time is permitted to elapse permitting the locatorto preferentially concentrate at any marker and for unbound locator tobe cleared from the body so as to increase the ratio offluorescing-based radiation from specifically bound locator tofluorescing-based radiation representing background in the patient. Ingeneral, the clearing time is determined by the form of antibody orlocator employed and typically will be from about two to about threeweeks. Background determinations can be made in conjunction with bloodtests. After the clearance time has elapsed, the patient is surgicallyassessed at an operative field. As the initial component of assessingthe field, the above-described squelch procedure is carried out. Such aprocedure was carried out during the era of RIGS where the radioisotopedetecting probe would be held in stationary position such as at theaorta and background blood pool radiation values were determined.Because of the vascularity of tissue, with the instant system, squelchis undertaken with a scanning.

FIG. 12 is a flow chart illustrating scan routines employed with thesystem. Looking to the figure, node 366 reappears in conjunction witharrow 370 and block 372. Block 372 provides for a prompt at display 130instructing the practitioner to remove the probe cap B or C. A furtherprompt instructing the practitioner to press the squelch button 180 maybe published. Then, as represented at arrow 374 and block 376, adetermination is made as to whether the squelch switch has beenactuated. If it has not, then as represented by loop line 378, thesystem dwells. Where the squelch switch has been actuated, then asrepresented at arrow 380 and block 382 the practitioner, using theprobe, scans over normal tissue for two seconds in repeating 0.1 secondsampling intervals. A resulting fluorescing intensity data is treated toderive a mean value and, as represented at arrow 384 and block 386, thatmean value is stored as SQUELCH LEVEL and the procedure continues asrepresented at arrow 388.

Looking momentarily to FIG. 13, a timing diagram corresponding with theutilization of a Type I nanocrystal-based locator is provided. Thediagram shows time in microseconds and modulation carried out with clockpulses as represented at diagram 400. In the figure, amplitudes arearbitrary and excitation pulses as well as enablement of the photodiodeare again arbitrarily established as one microsecond. In this regard,the excitation pulses are represented at diagram 402 and the enablementor driving of the photodiode are represented in general at 404. In thisregard, for a Type I nanocrystal, the photodiode is enabled insynchronization with the energization of the excitation components.Diagram level 406 in FIG. 13 represents initialization data obtainedwith cap A installed. In this regard, as shown at amplitude 408 dataobtained with cap A installed shows a filter test amplitude at 408 witha combined DARK CURRENT evaluation represented at dashed line level 410.That level 410 is obtained by enabling the photodiode as shown at 412and reading DARK CURRENT as shown at 414. At level 416 of FIG. 13,tissue scan data is presented, amplitudes 408 and 410 reappearing butadditionally, an amplitude is shown at 418 which represents a conditionwherein the excitation diodes are not energized but the photodiode isenabled or driven. This amplitude is categorized as “ambient amplitude”.In the course of utilization of the probe, the user may alter itsforward surface orientation with respect to the tissue being scanned.This may admit some ambient illumination including the earlier-describedsteady-state infrared illumination from the incandescent lighting of theoperating room. Note that the data representing amplitude 418 iscollected with the photodiode on as represented at 412 and theexcitation diodes are off. When the probe is scanning over normal orunaffected tissue an amplitude, for example, as shown at 420 may beencountered which will be considered to be statistically insignificant,for example, being less than three standard deviations over the meansquelch level developed as represented at block 386 in FIG. 12. Ineffect, amplitude 420 will represent detected fluorescence from whichthe sum of the leakage of filter 42 and any ambient illumination issubtracted. Amplitude 422 represents the positioning of the probeforward surface against a tissue location wherein the fluorescingradiation intensity is at a level which is statistically significant andthe system generates a perceptible cue. It will be beneficial toperiodically compare the level 418 of ambient illumination with SQUELCHLEVEL and alert the practitioner with respect to any ambiguity showing ahigher than anticipated ambient illumination level.

Returning to FIG. 12, arrow 388 is seen to be directed to block 430which provides for the reading of a data pulse and the subtraction ofthe ambient pulse, the initialization corrections having beenaccommodated for. Integration of the data pulse as corrected occurs in0.1 second intervals, to provide a fluorescing intensity level ascorrected data output which, as represented at arrow 432 and block 434is displayed at display 130. Where the user wishes to determine thedisplayed value, then the report switch 80 is momentarily depressed toevoke a voice transmission of the number representing fluorescingintensity. This data is treated by the control assembly as representedat arrow 436 and block 438 where an analysis is made as to whether thecorrected data is of a value representing three standard deviationsabove SQUELCH LEVEL. A three-sigma criterion for significance isselected because the expected probability of a false positive readingwould be less than 1% for such an analysis. Where that criteria ofstatistical significance is met, then as represented at arrow 438 andblock 440 the system generates a sound at loudspeaker 116 andilluminates light emitting diode 86. At this point of the procedure, thesurgeon may wish to depress the record switch 80 for an extendedinterval to activate a microphone and record the location of thislocator concentration. It also should be pointed out that the surgeongenerally will have removed readily discernable neoplastic tissue whichwill have been located by external imaging systems as well as byobservation and palpation. However, where lymph nodes are involved, suchdetection techniques are not available and essentially only anintra-operative probe-based system will locate the cancer involved lymphnodes.

From block 440 the procedure continues as represented at arrows 442 and444 to the query posed at block 446 determining whether the recordswitch 80 has been actuated. In the event that it has been actuated fora short interval or in a “click” manner, then as represented at arrow448 and block 450, the fluorescing intensity data is stored inconjunction with a time stamp. The procedure then continues asrepresented at arrows 452 and 454. Looking particularly to arrow 454where the record switch is held on by the surgeon, then a microphone isactivated and the surgeon may comment upon the activity then beingundertaken. For example, identifying the location of a removed lymphnode as represented at block 456 and verbal documentation is recorded.Arrow 458 extends from block 456 to the query at block 460 determiningwhether the power switch has been turned to an off position. In theevent that it has, then as represented at arrow 462 and block 464, alldefaults are reset and power is terminated. In the event of a negativedetermination at block 460, then as represented at arrow 466, theprocedure reverts to arrow 474.

Referring to FIG. 14, a timing diagram is provided concerning the use ofa Type II fluorescing nanocrystal and locator combination. In the FIG.,clock pulses are indicated at level 470, while excitation pulses arerepresented at level 472. In this regard, excitation is seen to occurfor a microsecond interval. The enabling or driving of photodiode 36 isrepresented at level 474. Such enablement occurs about 10 nanosecondsfollowing the termination of the excitation pulse as represented ingeneral at 476. It may be recalled that when a photon of excitation isabsorbed by a fluorescing nanocrystal of this type, the emitted photonhas a random delay time or time constant that is of the order of amicrosecond. Thus, if the modulation of the excitation has the form ofshort pulses, the fluorescence will continue after the excitation hasstopped. To use this effect and eliminate the effect at the photodiodeoccasioned by scattered excitation energy, the reading pulse is starteda short time after the excitation pulse has ended. Resultant tissuescanned data is represented at timing diagram level 478. At that level,for example, at reading interval 480 occasioned when the probe is overnormal tissue the data will represent a combination of ambientillumination and an exponential decay of fluorescence, it being recalledthat fluorescence also will occur during the excitation interval. Asbefore, ambient illumination is measured as shown at reading 482 whichoccurs with the photodiode in an enabled condition and with the absenceof excitation energy.

Where the frontal surface of the probe is over a statisticallysignificant region of locator, the exponential decay will be from ahigher level as represented at reading 484. Computation of the totalfluorescence intensity is computed by convolution procedure.

Probes employed with the instant system and method may be provided withconfigurations other than that shown and described in connection withFIGS. 5, 6, 6A and 7. One alternative approach is to configure the probesuch that it will be slideably positioned upon a finger of the surgeon.This is quite helpful in maintaining a proper orientation of the probewith respect to tissue being scanned, particularly in hard to accessareas. Referring to FIG. 16, a “finger probe” assembly is representedgenerally at 490 as it is carried by a surgeon represented schematicallyat 492. The probe component of the assembly 490 is shown at 494 beingslideably mounted on the second finger 496 of the hand 498 of surgeon490. Probe component 494 is represented as being in an active detectionposition such that it is mounted over the portion of finger 496corresponding with one phalanx bone (connected to the tip phalanx),although location beneath the glove has been contemplated. It may, ofcourse, be used on a different finger at the surgeon's discretion. As isapparent, the component 494 is located outwardly of the surgeon's gloveand further extending outwardly of the glove is an initial length ofcommunicating cable or wiring 500 which extends a limited distance alongthe forearm 502 of surgeon 490, whereupon it is secured by a strap 504in the vicinity of the elbow joint. It then is seen to extend to theshoulder region 506 of surgeon 490 at which position a circuit housing508 is fastened to the surgeon's arm, for example, by a strap orconnector 510. With the exception of switches 74 and 80, the housing 508may include the electronic components described in connection with FIG.4A. Additionally, a second pre-amplification stage may be incorporatedwithin circuit housing 508. From the circuit housing 508 a cable 510 asdescribed at 92 earlier herein may extend to a console as described at172 in FIG. 5.

In FIG. 15, the “back” side of probe 494, which is an elastomeric strap,is seen. Looking to FIG. 16, the opposite side of this probe component494 is revealed. Shown in FIG. 16 as being mounted at or near the tip ofthe underside of the finger 496, the length of probe 494 along finger496 is about 2 cm. This short length permits retention of the devicewhile allowing finger flexure. Probe component 494 carries non-imagingoptical components configured in the same manner described in connectionwith FIGS. 6, 6A and 7. Those components are represented in general inFIG. 16 at 514. Where the component 494 is not in active use, thesurgeon may simply rotate it about finger 496 to the top side of hand498.

Looking to FIG. 17, the probe component 494 is shown in perspective.Component 494 may be formed, for instance, from aluminum or plastic andis configured having a finger mount represented generally at 516 whichincludes a support region 518 and an oppositely disposed concave mountportion 520. Support portion 518 carries the probe working endnon-imaging optics including a transparent polycarbonate window, acylindrical copper excitation mount and heat sink, longpass filter,photodiodes and associated preamplifier. In the figure, the transparentwindow is seen at 522, covering the probe top, an array of eight lightemitting excitation diodes is represented in general at 524 and alongpass filter is represented at 526 behind window 522.

FIG. 17 reveals that the outward surface of the concave mount portion520 is configured in concave fashion to develop an elongate guideways528 and 530 extending along the lengthwise extent of the component 494.These guideways 528 and 530 aid in maintaining and changing theorientation of the probe component 494 through their abutting contactwith adjacent fingers of the surgeon's hand. The internal region of theconcave mount portion 520 is configured substantially as a halfcylindrical surface 532 of radius selected for nesting against thesurgeon's mounting finger. This surface 532 extends to two oppositelydisposed strap connector portions 534 and 536, each of which is formedas an elongate slot. These connector portions 534 and 536 serve toprovide for the attachment of an elastomeric web-like strap 538 whichfunctions to retain the finger mount against the surgeon's hand andexhibits sufficient flexure or elasticity so as to permit the movementof the probe component about the finger.

FIG. 18 looks to a sectional view of the probe 494 which includes theupper cap-shaped transparent polycarbonate window 522. Immediatelyadjacent the central portion of window 522 is longpass filter 526 whichextends over the outwardly facing surface of photodiode 542 which isoperationally combined with a preamplifier 544. Surrounding thisassemblage in the manner described in FIG. 6A is a cylindrical copperheat sink and excitation component support 546. The forward edge ofsupport 546 is canted inwardly to cause the excitation outputs of thearray of excitation light emitting diodes 524 to converge as representedby dashed lines 548 and 550. Additionally as in the case of FIGS. 6 and6A, the light emitting excitation diodes are mounted upon a thin Kapton(polyimide) carried printed circuit, the leads from which extend tocable 500 as well as the leads extending to and from pre-amplificationstage 544. Polymeric support material 552 is shown supporting thedetector components as well as transparent window cap 522.

A similar finger probe was developed for utilization with the RIGSsystem involving the detection of radioisotope. That implementation forthe RIGS system is described in detail in U.S. Pat. No. 5,441,050 byThurston and Olson, issued Aug. 15, 1995, entitled “Radiation ResponsiveSurgical Instrument” and incorporated herein by reference.

The probe detector components of the present system also may be adaptedto utilization in the course of laparoscopic surgery. From alaparoscopic surgical standpoint, it is necessary that the laparoscopicinstrument be maneuverable, having an access tube of a diameter limitedby the port of a cannula, for example, less than 12 mm. Experience withthe RIGS system has shown that the detection component of such aninstrument should be “side looking”. In this regard, the forwardtransmission surface of the detector components should be parallel withthe axis of the instrument. As the detection component of the instrumentis maneuvered within the insufflated body cavity, it is observed inreal-time two dimensionally with a television camera which also isinserted through a cannula into the body cavity.

Referring to FIG. 19, a laparoscopic instrument identified generally at560 is represented in perspective. The working end of instrument 560 isshown in general at 562. End 562 is coupled to a support which nowcomprises an elongate accessing tube 564 which, in turn, is coupled witha hand graspable handle 566. Extending from handle 566 is a cable againidentified at 92 which is coupled to a cable receptacle 174 of a console172 (FIG. 5). Accessing tube 564 will have a length of about 14 inches(36 cm) and a diameter of 11 mm permitting its insertion through a 12 mmdiameter cannula. The transmission surface of working end 562 is shownat 568. As described in connection with FIG. 4A, handle 566 mayincorporate a background or squelch switch 74; a record switch 80 and anLED 86 which functions as a cue representing the detection of astatistically significant amount of fluorescing radiation. As discussedearlier, that cue can be combined with an audible cue emanating from theconsole 172.

Looking to FIG. 20, working end 562 is revealed at an enhanced level ofdetail in conjunction with instrument axis 570. Detector structuring isseen to be quite similar to that described in connection with FIG. 6A.In this regard, a longpass filter is shown at 572 in immediate adjacencywith a transparent polycarbonate window 574, the outer surface of whichconstitutes a transmission surface. Immediately beneath the longpassfilter 568 is a photo-detector 576 which is combined with a preamplifier578. A polymeric support 580 surrounds and supports components 572, 576and 578 and provides a cylindrically shaped outer surface against whicha copper excitation component support and heatsink 582 is mounted. Theupwardly disposed edge of copper support 582 is canted inwardly and, inturn, supports a polyimide (Kapton) substrate carrying a printed circuitto which an array 584 of red region light emitting diodes (LEDs) ismounted. With such an arrangement, a converging red region excitationenergy is generated as represented at dashed lines 586 and 588. Lookingmomentarily to FIG. 21, the LED array 584 is revealed as it is presentbeneath transparent window 574. These excitation LEDs are identified at588 a-588 h. Recalling that the widthwise extent of the working end is11 mm, the arrangement illustrated in FIG. 21 is acceptable. The LEDs,for example, will have a rectangular ceramic substrate having a width of0.8 mm and a length of 1.6 mm.

Returning to FIG. 20, it may be observed that non-imaging opticalcomponents as well as preamplifier 578 are mounted upon a printedcircuit board 590 which, additionally, is coupled with multi-strandleads 592 and 594 extending to handle 566. The printed circuit boardassemblage in turn is mounted within a channel-shaped cradle 596 towhich polymeric spacers as identified at 598 are adhesively attached.The fluid-tight union between working end 562 and accessing tube 564 isshown at 600. In view of the length of accessing tube 564, asupplementary pre-amplification stage may be incorporated within handle566.

Transparent window 574 is seen to extend over the substantial flatportion of working end 562. Inasmuch as red excitation spectral energyis transmitted through it, a small portion of that energy will be lightpiped along it to enhance its visibility by a television camera.

The laparoscopic probe designed for utilization with the earlier RIGSsystem is described in U.S. Pat. No. 5,429,133 by Thurston, et al.,entitled “Radiation Responsive Laparoscopic Instrument”, issued Jul. 4,1995.

Since certain changes may be made in the above-described method, systemand apparatus without departing from the scope of the disclosure hereininvolved, it is intended that all matter contained in the descriptionthereof or shown in the accompanying drawings shall be interpreted asillustrative and not in a limiting sense.

1. A system for detecting fluorescing nanocrystal tracers at the body ofa patient, such nanocrystals being responsive to wavelength definedexcitation energy to fluoresce at one or more detection wavelengths,comprising: a probe having a working portion with a forward transmissionsurface, a photo-detector having a detector output corresponding withnon-imaged impinging fluorescent radiation when enabled, mounted inadjacency with said surface, a longpass filter operatively associatedwith the photo-detector and one or more excitation componentsenergizable to provide said excitation energy forwardly of saidtransmission surface, and a support for supporting said working portion;and a control assembly, responsive in an initialization mode to deriveand store a reference value not operationally associated with saidtracer, responsive in a range mode to energize said excitationcomponents under predetermined modulation and enable the photo-detectorwhile the transmission surface is adjacent tissue representingbackground to derive and store a mean squelch intensity levelcorresponding with said detection wavelengths and, when in a scan modewherein said transmission surface is moved in adjacency with tissuesuspected of being neoplastic, to energize the excitation componentsunder predetermined modulation and enable the photo-detector to derivethe detector output corresponding with said detection wavelengths withintensity levels minus said reference value as a corrected detectoroutput, and responsive to provide a humanly perceptible cue when saidcorrected detector output represents an intensity level statisticallysignificantly above said mean squelch intensity level.
 2. The system ofclaim 1 in which: said control assembly predetermined modulation isclock pulse-based.
 3. The system of claim 1 in which: said tracerincorporates a fluorescing nanocrystal and specifically binds a markerproduced by or associated with neoplastic tissue; said detectionwavelengths are in the near-infrared region; and said excitationcomponents comprise one or more light emitting diodes located adjacentsaid transmission surface in spaced adjacency with said photo-detector.4. The system of claim 3 in which: said light emitting diodes are cantedinwardly to an extent effecting a general convergence of their lightoutputs at a distance therefrom corresponding with the tissuepenetration capability of said wavelength defined excitation energy. 5.The system of claim 1 in which: said corrected detector output, whenrepresenting a statistically significant intensity level, exhibits anintensity level about three standard deviations above said mean squelchintensity level.
 6. The system of claim 1 in which: said probe supportis configured generally as a cylinder having a length effective for handgrasping.
 7. The system of claim 1 in which: said probe support isconfigured to be slideably retained upon a human finger.
 8. The systemof claim 1 in which: said probe is configured as a laparoscopicinstrument in which said probe support is generally an elongatecylindrical shaft extending along a shaft axis.
 9. The system of claim 8in which: said working portion forward transmission surface is within aplane generally parallel to said shaft axis.
 10. The system of claim 1in which: said tracer incorporates a first type fluorescing nanocrystalexhibiting a rapid fluorescing reaction response occurring substantiallyonly during its excitation and specifically binds a marker-produced byor associated with neoplastic tissue; and said control assemblypredetermined modulation is clock pulse-based, is responsive to anexcitation clock pulse to energize said excitation components for anexcitation interval and to simultaneously enable the photo-detector. 11.The system of claim 10 in which: said control assembly is responsive toa clock pulse occurring subsequent to said excitation clock pulse toenable the photo-detector in the absence of excitation energy anddetermine an ambient related value of detector output corresponding withany ambient illumination; and said corrected detector output is furtherderived with subtraction of said ambient related value.
 12. The systemof claim 1 in which: said tracer comprises a first type fluorescingnanocrystal; said tracer incorporates a second type fluorescingnanocrystal exhibiting decaying fluorescence at said detectionwavelength for a follow-on interval subsequent to the termination ofenergization of the excitation components, and specifically binds amarker; and said control assembly predetermined modulation is clockpulse-based, is responsive to an excitation clock pulse to energize saidexcitation components and enables the photo-detector for a samplinginterval subsequent to the termination of energization of the excitationcomponent.
 13. The system of claim 12 in which: said control assembly isresponsive to the intensities of said decaying fluorescence to computetotal fluorescing intensity occurring during both the energization ofthe excitation components and during the sampling interval.
 14. Thesystem of claim 13 in which: the computation of total fluorescingintensity is by convolution.
 15. The system of claim 12 in which: saidcontrol assembly is responsive to a clock pulse occurring subsequent tosaid sampling interval to enable the photo-detector in the absence ofexcitation energy and determine an ambient related value of detectoroutput corresponding with any ambient illumination; and said correcteddetector output is further derived with subtraction of saidambient-related value.
 16. The system of claim 1 further comprising: aninitialization chamber removably receptive of said probe forwardtransmission surface in light-tight relationship and having a polymericmaterial located forwardly of said surface exhibiting scatteringcharacteristics similar to those of tissue; said control assemblyinitialization mode is carried out with said transmission surface beinglocated within said chamber in light-tight relationship, and enablingsaid photo-detector to determine and store any electronic noise and darkcurrent value; said initialization mode being further carried out withsaid transmission surface being located within said chamber inlight-tight relationship and energizing said excitation components andenabling said photo-detector to determine and store an intensity valueof scattered excitation light; and said control assembly is responsiveto combine said electronic noise and dark current value with saidintensity value of scattered excitation light to provide said referencevalue.
 17. The system of claim 16 wherein: said tracer incorporates afirst type fluorescing nanocrystal, exhibiting a substantially immediatereaction response to excitation energy, and specifically binds a markerproduced by or associated with neoplastic tissue; and said controlassembly, when in said initialization mode, effects energization of saidexcitation components for an excitation interval while simultaneouslyenabling the photo-detector to derive said intensity value of scatteredexcitation light.
 18. The system of claim 16 wherein: said tracerincorporates a second type fluorescing nanocrystal exhibiting decayingfluorescence at said detection wavelength for a follow-on intervaloccurring subsequent to the termination of energization of theexcitation components, and specifically binds a marker produced by orassociated with neoplastic tissue; and said control assembly, when insaid initialization mode, effects energization of said excitationcomponents for an excitation interval, then enables the photo-detectorfor a sampling interval to derive said intensity value of scatteredexcitation light.
 19. The system of claim 1 further comprising: asquelch switch located at said probe support and/or the controlassembly, hand actuateable to derive and store said mean squelchintensity level.
 20. The system of claim 1 further comprising: a recordswitch located at said probe support and/or the control assembly, handactuateable for a short interval to effect recordation of the correcteddata output in a data file of said control assembly.
 21. The system ofclaim 20 in which: said record switch, when actuated for said shortinterval effects at said control assembly, a voice annunciation of saidcorrected data output.
 22. The system of claim 21 in which: said recordswitch when actuated for a recording interval greater than said shortinterval effects a voice recordation at said control assembly.